Methods for detecting Listeria monocytogenes

ABSTRACT

A method of detecting  Listeria monocytogenes . The method comprises providing a culture device with a selective culture medium and a detection article comprising a first indicator system. The selective culture medium facilitates the growth of  Listeria  microorganisms. When a  Listeria  microorganism is detected in a sample contacted with the culture medium, the detection article is contacted with the culture medium to detect  Listeria monocytogenes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.13/976,064, filed Jun. 26, 2013, now U.S. Pat. No. 9,273,340, which is aNational Stage application filed under 35 U.S.C. 371 based onPCT/2011/067149, filed 23 Dec. 2011, which claims the benefit of U.S.Provisional Patent Application No. 61/428,722, filed Dec. 30, 2010,which is incorporated herein by reference in its entirety

BACKGROUND

Testing samples of all food, beverages, and water for pathogenicmicroorganisms may not be practical because of the expense and becausepathogenic microorganisms are rarely found in, for example,properly-processed food. Therefore, tests for the presence of indicatormicroorganisms are routinely used to test food and water to determinethe likelihood of contamination with human pathogens. The presence ofone or more indicator microorganisms can be an indication of fecalcontamination, for example, and may indicate the potential presence of apathogenic microorganism.

Coliform bacteria (“coliforms”) represent one example of indicatormicroorganisms. The coliform group includes a number of genera (e.g.,Citrobacter, Enterobacter, Escherichia, Hafnia, Klebsiella, andSerratia) of rod-shaped Gram-negative bacteria that are found in largenumbers in the feces of warm-blooded animals and are characterized bytheir ability to ferment lactose to acid and gas by-products. Althoughmost coliforms are only associated with opportunistic infections inhumans, some coliform bacteria (e.g., E. coli O157:H7 or other ShigaToxin producing E. coli—STEC) are associated with a higher incidence ofmorbidity and mortality.

Members of the Enterobacteriaceae family of microorganisms representanother example of indicator microorganisms. In addition to the coliformbacteria, this family also includes a large number of other rod-shapedGram-negative bacteria. Like coliform bacteria, the presence ofEnterobacteriaceae microorganisms in a food or water sample can indicatethe presence of fecal contamination and, thus, the possible presence ofhuman pathogens (e.g., Salmonella enterica, Enteritidis, Salmonellaenterica Typhimurium, Shigella species, and Cronobacter species).

There is a need for efficient methods to test for the presence ofpathogenic microorganisms in a sample.

SUMMARY

In general, the invention is directed to a method for assessing themicrobiological content of a sample (e.g., a sample of food or water, anenvironmental sample). In particular, the inventive method can detectthe presence or absence of a target microorganism in a sample that hasbeen found to contain an indicator organism that can indicate thepresence of the target microorganism. The inventive method includesculturing a sample in a culture device with a first indicator system todetermine the presence of an indicator microorganism and, if anindicator microorganism is detected, contacting the culture device witha detection article comprising a second indicator system to determinethe presence or absence of a target microorganism.

In one aspect, the present disclosure provides a method of detecting thepresence or absence of a target microorganism. The method can compriseproviding a culture device including a culture medium comprisingingredients selected to facilitate growth of a predetermined indicatormicroorganism, a detection article comprising a first indicator system,and a sample. The first indicator system can be selected to detect atarget microorganism. The method further can comprise, inoculating theculture device with the sample, incubating the inoculated culture devicefor a period of time sufficient to permit growth of the indicatormicroorganisms, observing the culture device for an indication of apresence of at least one indicator microorganism, contacting the culturemedium of the incubated culture device with the detection article, andobserving the article-contacted culture device to detect a conversion ofthe first indicator system from a first state to a second state. In someembodiments, the conversion of the first indicator system from a firststate to a second state, if present, can be indicative of the presenceof at least one target microorganism. In some embodiments, theconversion of the first indicator system from a first state to a secondstate, if absent, is indicative of the presence of at least one targetmicroorganism.

In some embodiments, the method further can comprise providing a secondindicator system and placing the second indicator system in fluidcommunication with the culture medium, wherein observing the culturedevice for a presence of at least one indicator microorganism comprisesdetecting a presence or absence of a conversion of the second indicatorsystem from a first state to a second state. In any of the aboveembodiments, providing a culture device further can comprise providing aculture device that includes the second indicator system. In any of theabove embodiments of the method, providing the culture device furthercan comprise providing a culture device that comprises a hydrogel or adry, cold-water-soluble gelling agent.

In any of the above embodiments of the method, contacting the culturemedium with the detection article is performed only when the indicationof the presence of at least one indicator microorganism is observed.

In any of the above embodiments, providing a culture medium can compriseproviding a culture medium selected to facilitate growth of anEnterobacteriaceae microorganism, wherein providing a detection articlecan comprise providing a detection article to detect a microorganism ofthe genus Salmonella. In some embodiments, the first indicator systemcan comprise a reagent to detect α-galactosidase or caprylate esteraseenzyme activity.

In any of the above embodiments, providing a culture medium can compriseproviding a culture medium selected to facilitate growth of anEnterobacteriaceae microorganism, wherein providing a detection articlecan comprise providing a detection article to detect a microorganism ofthe genus Shigella. In some embodiments, the first indicator system cancomprise a reagent to detect β-glucosidase, β-fucosidase,N-acetyl-β-galactosaminidase, or a combination of any two or more of theforegoing enzyme activities.

In any of the above embodiments, providing a culture medium can compriseproviding a culture medium selected to facilitate growth of anEnterobacteriaceae microorganism, wherein providing a detection articlecan comprise providing a detection article to detect a microorganism ofthe genus Cronobacter. In some embodiments, the first indicator systemcan comprise a reagent to detect α-glucosidase and/or β-cellobiosidaseenzyme activity.

In any of the above embodiments, providing a culture medium can compriseproviding a culture medium selected to facilitate growth of anEnterobacteriaceae microorganism, wherein providing a detection articlecan comprise providing a detection article to detect Escherichia coli.In any of the above embodiments, providing a culture medium comprisesproviding a culture medium selected to facilitate growth of a coliformmicroorganism, wherein providing a detection article comprises providinga detection article to detect Escherichia coli. In some embodiments, thefirst indicator system can comprise a reagent to detect β-glucuronidaseenzyme activity.

In some embodiments, providing a culture medium can comprise providing aculture medium selected to facilitate growth of a microorganisms of theListeria genus, wherein providing a detection article can compriseproviding a detection article to detect Listeria monocytogenes. In someembodiments, the first indicator system can comprise a reagent to detectα-mannopyranosidase or phosphatidylinositol-specific phospholipase Cenzyme activity.

In any of the above embodiments, observing the culture device or thearticle-contacted culture device can comprise observing the culturedevice visually. In any of the above embodiments, observing the culturedevice or the article-contacted culture device can comprise observingthe culture device using an automated reader. In any of the aboveembodiments, the method further can comprise enumerating a portion ofindicator microorganism colony-forming units. In any of the aboveembodiments, the method further can comprise enumerating a portion oftarget microorganism colony-forming units.

In any of the above embodiments, contacting the culture medium with thedetection article further can comprise contacting the culture medium ata predetermined temperature.

In another aspect, the present disclosure provides an article. Thearticle can comprise a substrate with upper and lower major surfaces anda coating disposed on at least one of the major surfaces. The coatingcan comprise a first indicator system. The indicator system can beconverted from a first state to a second state by α-galactopyranoside orcaprylate esterase enzyme activity. In some embodiments of the article,the first indicator system can be selected from the group consisting of5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside,5-bromo-6-chloro-3-indolyl-caprylic acid, and5-bromo-4-chloro-3-indolyl-caprylic acid.

In yet another aspect, the present disclosure provides an article. Thearticle can comprise a substrate with upper and lower major surfaces anda coating disposed on at least one of the major surfaces. The coatingcan comprise a first indicator system. The first indicator system can beconverted from a first state to a second state by β-glucuronidase enzymeactivity. In some embodiments, the first indicator system can beselected from the group consisting of5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid,p-nitrophenyl-β-glucuronide,p-nitrophenyl-2,3,4-tri-O-acetyl-β-glucuronic acid methyl ester,phenolphthalein glucuronic acid, phenolphthalein mono-P-glucuronic acid,naphthyl-AS-BI-β-D-glucuronide, and 4-methylumbelliferylβ-D-glucuronide, 8-Hydroxyquinoline-beta-D-glucuronic acid, sodium salt,2-Naphthyl-beta-D-glucuronic acid, sodium salt,4-Nitrophenyl-beta-D-glucuronic acid, sodium salt,Phenolphthalein-beta-D-glucuronic acid, sodium salt monohydrate,5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, cyclohexylammoniumsalt, 3-Indoxyl-beta-D-glucuronic acid, cyclohexylammonium salt,3-Indoxyl-beta-D-glucuronic acid, sodium salt,5-Bromo-6-chloro-3-indoxyl-beta-D-glucuronic acid, cyclohexylammoniumsalt, 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, sodium saltanhydrous, and 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, sodiumsalt trihydrate.

In yet another aspect, the present disclosure provides an article. Thearticle can comprise a substrate with upper and lower major surfaces anda coating disposed on at least one of the major surfaces. The coatingcan comprise a first indicator system. The first indicator system can beconverted from a first state to a second state by α-mannopyranosidase orphosphatidylinositol-specific phospholipase C enzyme activity. In someembodiments, the first indicator system can be selected from the groupconsisting of 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate,5-Bromo-6-chloro-3-indoxyl myo-inositol-1-phosphate, ammonium salt,4-Methylumbelliferyl myo-inositol-1-phosphate, N-methyl-morpholine salt,3-indoxyl-α-D-mannopyranoside, 5-bromo-3-indoxyl-α-D-mannopyranoside,4-chloro-3-indoxyl-α-D-mannopyranoside,5-iodo-3-indoxyl-α-D-mannopyranoside,5-bromo-4-chloro-3-indoxyl-α-D-mannopyranoside,chloro-3-indoxyl-α-D-mannopyranoside,5-bromo-6-chloro-3-indoxyl-α-D-mannopyranoside,6-bromo-3-indoxyl-α-D-mannopyranoside,6-chloro-3-indoxyl-α-D-mannopyranoside,6-fluoro-3-indoxyl-α-D-mannopyranoside,4,6-dichloro-3-indoxyl-α-D-mannopyranoside,6,7-dichloro-3-indoxyl-α-D-mannopyranoside,4,6,7-trichloro-3-indoxyl-α-D-mannopyranoside,5-bromo-4-chloro-N-methyl-α-D-mannopyranoside,3-indoxyl-α-D-mannopyranoside, andN-methyl-3-indoxy-α-D-mannopyranoside,6-Bromo-2-naphthyl-α-D-mannopyranoside,4-Methylumbelliferyl-α-D-mannopyranoside, and4-Nitrophenyl-α-D-mannopyranoside.

In any of the above embodiments, the coating can be disposed on theupper and lower major surfaces. In any of the above embodiments, thearticle further can comprise an adhesive layer. In some embodiments, atleast a portion of the first indicator system can be disposed on or inthe adhesive layer. In any of the above embodiments, the first indicatorsystem can be disposed on both major surfaces. In any of the aboveembodiments, the substrate can be selected from the group consisting ofa polymeric film, paper, a nonwoven, a membrane filter and derivativesof any of the foregoing. In any of the above embodiments, the coatingfurther can comprise a binder.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

As used herein, “a,” “an,” “the,” “at least one,” and “one or more” areused interchangeably. Thus, for example, a microorganism can beinterpreted to mean “one or more” microorganisms.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

“Culture device”, as used herein, refers to an article adapted to housea nutrient medium that facilitates the growth of a microorganism.Optionally, the culture device may comprise a lid or cover to minimizethe exposure of the nutrient medium to external contamination and/or toreduce the loss of moisture from the culture medium during incubationand/or storage. Nonlimiting examples of culture devices include flasks,beakers, tubes, Petri dishes, multi-well plates, PETRIFILM plates,COMPACT DRY media sheets, SANITA-KUN sheets, and the like.

“Indicator system”, as used herein, refers to one or more of any of thefollowing and any combination of one or more of the following: achromogenic enzyme substrate, a fluorogenic enzyme substrate, a redoxindicator (e.g., triphenyltetrazolium chloride, methylene blue), ametabolizable nutrient, pH indicator. “Metabolizable nutrient” refers toany molecule that can be used by a predetermined indicator organismand/or a predetermined target microorganism to produce biomass and/orenergy. The use of the metabolizable nutrient by the microorganismsdirectly or indirectly results in a pH or other detectable ionic changein an aqueous medium that is in fluid contact with the microorganism. A“differentiating” indicator system is an indicator system that can beused to distinguish two nonidentical microorganisms based on theirrespective reactivities with a component(s) of the indicator system(s).

“Indicator microorganism”, as used herein, refers to a microorganismthat belongs to a group of microorganisms that is known to be found inan environment (e.g. a liquid or solid matrix) in which a targetmicroorganism is also found. According to the present disclosure,indicator microorganisms and their corresponding target microorganismsare capable of being cultured in the same culture medium. In addition,target microorganisms may react with the same indicator system thatdetects the corresponding indicator microorganisms. Indicatormicroorganisms can include relatively large and diverse groups ofmicroorganisms (e.g., aerobic bacteria, yeast, filamentous fungi),relatively large and relatively less diverse groups of microorganisms(e.g., a phylogenetically-related group of microorganisms such as theEnterobacteriaceae family, for example, or a physiologically-relatedgroup of microorganisms such as the coliform bacteria, for example), andeven smaller groups and/or relatively less diverse groups ofmicroorganisms (e.g., a genus such as Listeria, a species such asEscherichia coli).

“Target microorganism”, as used herein, refers a predeterminedmicroorganism that can be found in one or more of the same environmentsthat the indicator microorganisms are found. A target microorganism canbe distinguished from one or more of a group of indicator microorganismson the basis of its reactivity, or lack thereof, with a differentiatingindicator system. In some embodiments, the target microorganism canbelong to the group of indicator microorganisms (e.g., an exemplarytarget microorganism, Escherichia coli, is a member of theEnterobacteriaceae family, which is a group of indicator microorganismsknown in the art). In certain embodiments, “target microorganism” mayrefer to one or more strains of a particular species, one or morespecies of a particular genus, or more than one species from each of twoor more genera or one or more strains or species of a non-taxonomic(e.g., physiologically-related) group.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

Additional details of these and other embodiments are set forth in theaccompanying drawings and the description below. Other features, objectsand advantages will become apparent from the description and drawings,and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a block diagram of one embodiment of a method for detecting atarget microorganism according to the present disclosure.

FIG. 2 is a top view of one embodiment of a culture device with coloniesof indicator microorganisms disposed therein.

FIG. 3 is a perspective view of one embodiment of a detection articleaccording to the present disclosure.

FIG. 4 is a top view of the culture device of FIG. 3 after the placementof a detection article therein.

DETAILED DESCRIPTION

The invention is directed to a method for assessing the microbiologicalcontent of a sample. In particular, the method includes sequentialdetection processes that, advantageously, are conducted in a singleculture device. The detection processes are distinct, but related in thesense that the first detection process identifies a group of indicatormicroorganisms and the second detection process identifies a targetmicroorganism within the group. Advantageously, the result of the firstidentification process can be used to decide whether the use of thesecond detection process is indicated. Accordingly, in some embodiments,when an indicator microorganism is not detected by the firstidentification process, an operator can avoid the time, materials,labor, and expense of the second identification process.

FIG. 1 is a block diagram showing one embodiment of a method ofdetecting a target microorganism according to the present disclosure.Methods of the present disclosure are directed to testing a sample forthe presence or absence of a target microorganism. The method includesthe step 152 of providing a sample to be tested, a detection article,and a culture device that includes a culture medium. Optionally, asecond indicator system can be provided. The method further comprisesthe step 154 of placing the second indicator system, if present, influid communication with the culture medium. The method furthercomprises the step 156 of inoculating the culture device with thesample, the step 158 of incubating the inoculated culture device, andthe step 160 of observing the culture device for an indication of anindicator microorganism. If an indicator microorganism is observed instep 160, the method further comprises the optional step 162 ofcontacting the culture medium with the detection article and the step164 of observing the first indicator system. Each of the steps in themethod is described in greater detail below. An advantage of the methodis that the detection article may only need to be applied to the culturemedium if an indication of an indicator microorganism is detected. Thus,in samples where no indication of an indicator microorganism isdetected, it may be inferred that a target microorganism is not presentand the detection article does not have to be used.

Providing a sample to be tested may comprise providing a sample that issuspected of containing a target microorganism. The sample can be anysample that may include a target microorganism as defined herein.Nonlimiting examples of suitable samples include environmental samples(e.g., surface swabs/sponges, soil, sediments, fomites), food (e.g., rawmaterials, in-process samples, and finished-product samples), beverages,clinical/veterinary samples (e.g., blood, serum, plasma, urine, sputum,tissue, mucous, feces, wound exudate, pus, cerebrospinal fluid), andwater (e.g., surface water, potable water, process water).

In some embodiments, the presence or absence of a target microorganismcan be analyzed in a test sample that is derived from a variety of food,beverage, or food- or beverage-processing environmental sources.Non-limiting examples of food sources include raw or processed meat, rawor processed fruits or vegetables, non-fluid dairy products (e.g.,cheese, butter, and ice cream), nuts, spices, ingredients, and syrups.Non-limiting examples of beverage sources include potable water, fruitor vegetable juices, milk, and fermented beverages. Pasteurized food orbeverages may also be suitable sources. Non-limiting examples of food-or beverage-processing environmental samples include food-handlingsurface samples (e.g., conveyor belts, blades, cutting surfaces, mixingequipment surfaces, filters, storage containers), room samples (e.g.,walls, floors, drains, ventilation equipment), and cleaning equipment(e.g., hoses, cleaning tools).

In some embodiments, the presence or absence of a target microorganismcan be analyzed in a sample that is derived from a variety of human oranimal sources, such as a physiological fluid, e.g., blood, saliva,ocular lens fluid, synovial fluid, cerebral spinal fluid, pus, sweat,exudate, urine, mucus, lactation milk, or the like. Further, the testsample may be derived from a body site, e.g., wound, skin, nares, scalp,nails, etc.

Samples of particular interest from human or animal sources includemucus-containing samples, such as nasal samples (from, e.g., anterialnares, nasopharyngeal cavity, nasal cavities, anterior nasal vestibule,etc.), as well as samples from the outer ear, middle ear, mouth, rectum,vagina, or other similar tissue. Examples of specific mucosal tissuesinclude buccal, gingival, nasal, ocular, tracheal, bronchial,gastrointestinal, rectal, urethral, ureteral, vaginal, cervical, anduterine mucosal membranes.

Besides physiological fluids, other test samples may include otherliquids as well as solid(s) dissolved in a liquid medium. Samples ofinterest may include process streams, water, soil, plants or othervegetation, air, surfaces (e.g., contaminated surfaces), and the like.Samples can also include cultured cells. Samples can also includesamples on or in a device comprising cells, spores, or enzymes (e.g., abiological indicator device).

Suitable samples for methods of the present disclosure can includecertain solid samples. Solid samples may be disintegrated (e.g., byblending, sonication, homogenization) and may be suspended in a liquid(e.g., water, buffer, broth). In some embodiments, a sample-collectiondevice (e.g., a swab, a sponge) containing sample material may be usedin the method. Alternatively, the sample material may be eluted (e.g.,rinsed, scraped, expressed) from the sample-collection device beforeusing the sample material in the method. In some embodiments, liquid orsolid samples may be diluted in a liquid (e.g., water, buffer, broth).

The sample may comprise an indicator microorganism, as described herein.The indicator microorganism can be indicative of contamination (e.g.,fecal contamination), infection (e.g., infection with a pathogenicmicroorganism), or an indicator of general sanitation (e.g., any aerobicmicroorganism). The indicator microorganism further can be a targetmicroorganism.

Microorganisms of particular interest, which may be of interest as anindicator organism or a target microorganism, include prokaryotic andeukaryotic organisms, particularly Gram positive bacteria, Gram negativebacteria, fungi, mycoplasma, and yeast. Particularly relevant organismsinclude members of the family Enterobacteriaceae, or the familyMicrococcaceae or the genera Staphylococcus spp., Streptococcus spp.,Pseudomonas spp., Enterococcus spp., Salmonella spp., Legionella spp.,Shigella spp. Yersinia spp., Enterobacter spp., Escherichia spp.,Bacillus spp., Listeria spp., Vibrio spp., Corynebacteria spp. as wellas herpes virus, Aspergillus spp., Fusarium spp., and Candida spp.Particularly virulent organisms include Staphylococcus aureus (includingresistant strains such as Methicillin Resistant Staphylococcus aureus(MRSA)), S. epidermidis, Streptococcus pneumoniae, S. agalactiae, S.pyogenes, Enterococcus faecalis, Vancomycin Resistant Enterococcus(VRE), Vancomycin Resistant Staphylococcus aureus (VRSA), VancomycinIntermediate-resistant Staphylococcus aureus (VISA), Bacillus anthracis,Pseudomonas aeruginosa, Escherichia coli, Aspergillus niger, A.fumigatus, A. clavatus, Fusarium solani, F. oxysporum, F.chlamydosporum, Listeria monocytogenes, Listeria ivanovii, Vibriocholera, V parahemolyticus, Salmonella choleraesuis, S. typhi, S.typhimurium, Candida albicans, C. glabrata, C. krusei, Cronobactersakazakii, E. coli O157 and multiple drug resistant Gram negative rods(MDR).

Gram positive and Gram negative bacteria are of particular interest. Ofparticular interest are Gram positive bacteria, such as Listeriamonocytogenes. Also, of particular interest are antibiotic resistantmicrobes including MRSA, VRSA, VISA, VRE, and MDR microbes.

Referring back to step 152 of FIG. 1, in addition to providing a sampleto be tested, the method further includes providing a culture device, afirst indicator system, and an article with a second indicator systemcoated thereon. Culture device is used in a broad sense and includes avariety of articles adapted to house a nutrient medium that facilitatesthe growth of a microorganism. The selection of a particular nutrientmedium to facilitate the growth of any particular indicatormicroorganism according to the method is within the grasp of a personhaving ordinary skill in the art.

In some embodiments, the nutrient medium can comprise one or moreselective inhibitors. “Selective inhibitors”, as used herein, refers tochemical compounds that are added to nutrient medium to partially orcompletely inhibit the growth of certain susceptible microorganisms orgroups of microorganisms, thereby selectively favoring the growth ofother microorganisms or groups of microorganisms. Selective inhibitorsare known in the art and include, for example, bile salts, inorganicsalts (e.g., NaCl, LiCl, MgCl₂), and antibiotics (fluoroquinolones,β-lactam antibiotics, aminoglycosides). In certain preferred embodimentsof the present disclosure, the nutrient medium can include selectiveinhibitors at concentrations that are minimally-selective (i.e., atconcentrations that are below the customary concentrations used inselective growth media). Advantageously, the use of minimally-selectivenutrient medium can permit the recovery and detection of more targetmicroorganisms using the method of the present disclosure. Without beingbound by theory, this may be possible because the lower concentration ofselective inhibitors permit the growth of injured, stressed, and/orrelatively-susceptible target microorganisms in the selective medium,thereby enabling their detection with the second indicator system.

The first indicator system is used to indicate the presence or absenceof a target microorganism. The optional second indicator system is usedto indicate the presence or absence of an indicator microorganism. Thefirst and/or second indicator system can comprise a chromogenic enzymesubstrate, a fluorogenic enzyme substrate, a redox indicator, ametabolizable nutrient, a pH indicator, or any combination of two ormore of the foregoing. In some embodiments, the combination of a pHindicator and a certain metabolizable nutrient may be provided for themethod by providing one of the components in the culture medium of theculture device and by providing the other component in the detectionarticle.

The choice of the first indicator system may depend upon the targetmicroorganism and/or the culture medium used in the culture device andsuch choices are guided by the present disclosure, as will be recognizedby a person having ordinary skill in the art. In some embodiments of themethod, a highly-differential first indicator system (i.e., an indicatorsystem that reacts with relatively few microorganisms, including thetarget microorganism) can be used in conjunction with a minimallyselective culture medium. In these embodiments, the minimally-selectiveculture medium can permit the recovery and growth of injured and/orstressed target microorganisms, thereby enabling the detection of suchtarget organisms that may be inhibited by a relatively highly-selectiveculture medium.

In some embodiments of the method, a relatively less-differential firstindicator system (i.e., an indicator system that reacts with relativelymany microorganisms, including the target microorganism) can be used inconjunction with a relatively highly-selective culture medium. Thisapproach may be used with highly complex samples (e.g., samples thatrequire strongly-selective conditions such as samples from floor drainsthat tend to have large-highly diverse microbial contents or samplesfrom relatively non-selective pre-enrichment broth cultures). An exampleof this approach is the use of the Demi-Fraser/UVM, Fraser brothenrichment system with Modified Oxford agar detection of Listeriamicroorganisms.

In any of the embodiments, the first or second indicator system cancomprise an oxidation-reduction indicator (also called a redoxindicator) suitable to biological oxidation-reduction reactions.Oxidation-reduction indicator dyes may be pH-dependent orpH-independent. Nonlimiting examples of oxidation-reduction indicatordyes include 2,2′-Bipyridine (Ru complex), Nitrophenanthroline (Fecomplex), N-Phenylanthranilic acid, 1,10-Phenanthroline (Fe complex),N-Ethoxychrysoidine, 2,2′-Bipyridine (Fe complex),5,6-Dimethylphenanthroline (Fe complex), o-Dianisidine, Sodiumdiphenylamine sulfonate, Diphenylbenzidine, Diphenylamine, Viologen,Sodium 2,6-Dibromophenol-indophenol, Sodium2,6-Dichlorophenol-indophenol, Sodium o-Cresol indophenol, Thionine(syn. Lauth's violet), Methylene blue, Indigotetrasulfonic acid,Indigotrisulfonic acid, Indigodisulfonic acid, Indigomonosulfonic acid,Phenosafranin, Safranin T, and Neutral red.

In any of the embodiments, the first and/or second indicator system cancomprise a chromogenic enzyme substrate. Eligible chromogenic enzymesubstrates include bromo-chloro-indolyl derivatives, nitrophenylderivatives, and phenolphthalein derivatives, for example.

Useful 5-bromo-4-chloro-3-indolyl derivatives include5-bromo-6-chloro-3-indolyl acetate, 5-bromo-4-chloro-3-indolyl acetate,5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside,5-bromo-4-chloro-3-indoyl-1,3-diacetate, 5bromo-4-chloro-3-indolyl-β-D-fucopyranoside,5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside,5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid,5-bromo-4-chloro-3-indolyl phosphate, and 5-bromo-4-chloro-3-indolylsulfate.

Useful nitrophenyl derivatives include p-nitrophenol and o-nitrophenolderivatives. Particularly useful p-nitrophenols includediethyl-p-nitrophenyl phosphate; di-pnitrophenyl phosphate;p-nitrophenyl-2-acetamido-2-deoxy-3-O-β-galactopyranosyl-β-glucopyranoside;p-nitrophenyl-2-acetamido-2-deoxy-β-glucopyranoside;p-nitrophenylacetate, p-nitrophenyl-N-acetyl-β-D-glucosaminide,p-nitrophenyl-β-D-N,N′-diacetylchitobioside;p-nitrophenyl-α-glucopyranoside, p-nitrophenyl-α-maltoside;p-nitrophenyl-β-maltoside; p-nitrophenyl-α-mannopyranoside;p-nitrophenyl-β-mannopyranoside; p-nitrophenyl myristate; p-nitrophenylpalmitate; p-nitrophenyl phosphate; bis(p-nitrophenyl)phosphate;tris(p-nitrophenyl)phosphate; p-nitrophenyl-β-glucopyranoside;p-nitrophenyl-β-glucuronide; α-p-nitrophenylglycerine;p-nitrophenyl-α-rhamnopyranoside; p-nitrophenyl stearate; p-nitrophenylsulfate; p-nitrophenyl-2,3,4-tri-O-acetyl-β-glucuronic acid methylester; and p-nitrophenyl valerate.

Particularly useful o-nitrophenols include o-nitrophenyl acetate,o-nitrophenyl-β-glucoside and o-nitrophenyl-β-D-glucopyranoside. Otherparticularly useful nitrophenyl derivatives includenitrophenyl-β-fucopyranoside, nitrophenyl-o-galactopyranoside,nitrophenyl butyrate, nitrophenyl caprate, nitrophenyl caproate,nitrophenyl caprylate, nitrophenyl laurate, and nitrophenyl propionate.

Useful indoxyl derivatives include indoxyl-acetate; indoxylβ-D-glucoside; 3-indoxyl sulfate; 3-indoxyl phosphate.

Useful phenolphthalein derivatives include: phenolphthalein dibutyrate;phenolphthalein diphosphate; phenolphthalein disulfate; phenolphthaleinglucuronic acid; phenolphthalein mono-P-glucosiduronic acid;phenolphthalein mono-P-glucuronic acid; and phenolphthaleinmonophosphate.

All of the above-described chromogenic substrates will react directlywith an appropriate enzyme to produce a chromophore.

Additional enzyme substrates containing 1-naphthyl, 2-naphthyl andNaphthyl-AS-BI derivatives are usefully employed if the derivativeenzyme modified product is further reacted with a chromogenic reagent,such as diazotized dyes, e.g.,1-diazo-4-benzoylamino-2,5,diethoxybenzene, (commercially available as“Fast Blue BB Salt” from Sigma Chemical),1-diazo-4-benzoylamino-2,5-diethoxybenzene,p-diazo-2,5-diethoxy-N-benzoylalanine, chloro-2-methylbenzene diazoniumchloride, and o-aminoazotoluene diazonium salt, to produce achromophore.

Particularly useful 1-naphthyl derivatives include1-naphthyl-N-acetyl-β-D-glucosaminide.

Particularly useful 2-naphthyl derivatives include 2-naphthyl-phosphate;2-naphthyl-butyrate; 2-naphthyl-caprylate; 2-naphthyl-myristate;L-leucyl-2-naphthyla-mide; L-valyl-2-naphthylamide;L-cystyl-2-naphthylamide; N-benzoyl-DL-arginine-2-naphthylamide;N-glutaryl-phenylalanine 2-naphthylamine; 2-naphthyl-phosphate;6-Br-2-naphthyl-α-D-galactopyranoside; 2-naphthyl-β-D-galactopyranoside;2-naphthyl-2-D-glucopyranoside; 6-bromo-2-naphthol-β-D-glucopyranoside;6-bromo-2-naphthyl-2-D-mannopyranoside; and2-naphthyl-α-L-fucopyranoside.

Particularly useful naphthyl-AS-BI derivatives includenaphthyl-AS-BI-phosphate and naphthyl-AS-BI-β-D-glucuronide.

Where the enzyme whose activity is to be detected isalpha-D-glucosidase, a suitable chromogenic enzyme substrate, forexample, is p-nitrophenyl-α-glucopyranoside. Where the enzyme activityto be detected is alpha-L-arabinofuranosidase, a suitable chromogenicenzyme substrate, for example, is p-nitrophenyl-α-L-arabinofuranoside.Where the enzyme activity to be detected is beta-D-glucosidase, asuitable chromogenic enzyme substrate, for example, isp-nitrophenyl-β-D-glucopyranoside.

In any of the embodiments, the first and/or second indicator system cancomprise a fluorogenic enzyme substrate. Eligible fluorogenic enzymesubstrates include derivatives of 4-methylumbelliferone7-amido-4-methylcoumarin, fluorescein, rhodamine, and fluorescamine, forexample.

Suitable 4-methylumbelliferyl derivatives include, for example:4-methylumbelliferyl-2-acetamido-4,6-O-benzylidene-2-deoxy-β-D-glucopyranoside; 4-methylumbelliferylacetate; 4-methylumbelliferyl-N-acetyl-β-D-galactosaminide;4-methylumbelliferyl-N-acetyl-α-D-glucosaminide;4-methylumbelliferyl-N-acetyl-β-D-glucosaminide;2′-(4-methylumbelliferyl)-α-D-N-acetyl neuraminic acid;4-methylumbelliferyl α-L-arabinofuranoside; 4-methylumbelliferylα-L-arabinoside; 4-methylumbelliferyl butyrate; 4-methylumbelliferylβ-D-cellobioside; methylumbelliferyl β-D-N, N′-diacetylchitobioside; 4methylumbelliferyl elaidate; 4-methylumbelliferyl β-D-fucoside;4-methylumbelliferyl α-L-fucoside; 4-methylumbelliferyl β-L-fucoside;4-methylumbelliferyl α-D-galactoside; 4-methylumbelliferylβ-D-galactoside; 4-methylumbelliferyl α-D-glucoside;4-methylumbelliferyl β-D-glucoside; 4-methylumbelliferylβ-D-glucuronide; 4-methylumbelliferyl p-guanidinobenzoate;4-methylumbelliferyl heptanoate; 4-methylumbelliferylα-D-mannopyranoside; 4-methylumbelliferyl β-D-mannopyranoside;4-methylumbelliferyl oleate; 4-methylumbelliferyl palmitate;4-methylumbelliferyl phosphate; 4-methylumbelliferyl propionate;4-methylumbelliferyl stearate; 4-methylumbelliferyl sulfate;4-methylumbelliferyl β-D-N, N′, N″-triacetylchitotriose;4-methylumbelliferyl 2,3,5-tri-o-benzoyl-α-L-arabinofuranoside;4-methylumbelliferyl-p-trimethylammonium cinnamate chloride; and4-methylumbelliferyl β-D-xyloside.

Suitable 7-amido-4-methylcoumarin derivatives include, for example:L-alanine-7-amido-4-methylcoumarin; L-proline 7-amido-4-methylcoumarin;L-tyrosine-7-amido-4-methylcoumarin; L-leucine-7-amido-4-methylcoumarin;L-phenylalanine-7-amido-4-methylcoumarin; and7-glutarylphenylalanine-7-amido-4-methylcoumarin.

Suitable peptide derivatives of 7-amido-4-methyl coumarin include, forexample: N-t-BOC-Ile-Glu-Gly-Arg 7-amido-4-methylcoumarin;N-t-BOC-Leu-Ser-Thr-Arg 7-amido-4-methylcoumarin; N-CBZ-Phe-Arg7-amido-4-methyl-coumarin; Pro-Phe-Arg 7-amido-4-methylcoumarin;N-t-BOC-Val-Pro-Arg 7-amido-4-methylcoumarin; and N-glutaryl-Gly-Arg7-amido-4-methylcoumarin.

Suitable diacetylfluorescein derivatives include, for example,fluorescein diacetate, fluorescein di-(β-D-galactopyranoside), andfluorescein dilaurate.

Where the biological activity to be detected is alpha-D-glucosidase,chymotrypsin, suitable fluorogenic enzyme substrates are4-methylumbelliferyl-alpha-D-glucoside,7-glutarylphenylalanine-7-amido-4-methyl coumarin, or4-methylumbelliferyl heptanoate, respectively. Where the biologicalactivity to be detected is alpha-L-arabinofuranosidase, a suitablefluorogenic enzyme substrate is4-methylumbelliferyl-alpha-L-arabinofuranoside. Where the biologicalactivity to be detected is beta-D-glucosidase, a suitable fluorogenicenzyme substrate is 4-methylumbelliferyl-beta-D-glucoside.

In any of the embodiments of the method, the first and/or secondindicator system can comprise a pH indicator dye used in conjunctionwith a metabolizable nutrient. The pH indicator dye can be selectedaccording to criteria known in the art such as, for example, pH range,compatibility with the indicator and/or target microorganisms, andsolubility. In some embodiments, a salt form of the pH indicator may beused, for example, to increase the solubility of the pH indicator in anaqueous mixture. Nonlimiting examples of suitable pH indicator dyesinclude, for example, thymol blue, tropeolin 00, methyl yellow, methylorange, bromophenol blue, bromocresol green, methyl red, bromothymolblue, phenol red, neutral red, phenolphthalein, thymolphthalein,alizarin yellow, tropeolin O, nitramine, trinitrobenzoic acid, thymolblue, bromophenol bromphenol blue, tetrabromophenol blue, bromocresolgreen, bromocresol purple, methyl red, bromothymol blue, phenol red,Congo red, and cresol red.

The metabolizable nutrient can be any metabolizable nutrient known inthe art to react with at least one microorganism (either an indicatormicroorganism and/or a target microorganism) and to result in a pHchange (e.g., a localized pH change) in an aqueous medium that is influid contact with the microorganism. The nutrient can be selected froma variety of nutrient types known in the art. Non-limiting examples ofnutrient types include carbohydrates (e.g., sugars, polysaccharides, andderivatives thereof), fats (e.g., fatty acids, fatty acid esters, andderivatives thereof), amines (e.g., amino acids, peptides,oligopeptides, proteins, polyamines, and derivatives thereof),polyphosphates, purines, pyrimidines, nucleosides, and nucleotides.

In some embodiments, the optional second indicator system can beprovided in a culture device (e.g., in an agar culture medium in a petridish; in a dehydrated culture medium in a device such as a Petrifilmculture device, for example). When provided as a component of ahydrogel, such as a component of an agar culture medium, the optionalsecond indicator system is provided in fluid communication with theculture medium. When provided as a dehydrated component as part of arehydratable culture device, such as a Petrifilm plate, the optionalsecond indicator system is brought into fluid communication with theculture medium, as indicated in step 154 of FIG. 1. This can beaccomplished, for example, by rehydrating the dehydrated culture mediumin the device with a liquid (e.g., water, a buffer, a diluent). In someembodiments, rehydratable culture media include cold-water-solublegelling agents such as agar, agarose, guar gum, xanthan gum, locust beangum, polyvinyl alcohol, and/or polyvinylpyrrolidone, for example.Optionally, the liquid may contain the sample material, therebypermitting the simultaneous performance of step 154 and the inoculationstep 156 by the operator.

Inoculating the culture device can be done by a variety of methods thatare known in the art. Nonlimiting examples of suitable inoculationmethods include pour-plate techniques, surface inoculation techniques,streak-plating techniques, swab-plating techniques, and surfacecontact-plating techniques (e.g., Rodac plating methods). Filtermembrane plating techniques may be used in the present method, providedthat the membrane filter does not substantially interfere with thereaction between the microorganisms and the indicator systems orinterfere with the observation of the indicator systems.

Methods of the present disclosure include incubating an inoculatedculture device for a period of time. A person of ordinary skill in therelevant art will recognize that the incubation temperature may beselected according to the microorganism to be detected. For example, ifa yeast or mold is to be detected, the first incubation temperaturetypically may be from about room temperature (ca. 23° C.) to about 32°C. For example, if a bacterium is to be detected, the first incubationtemperature typically may be from about room temperature to about 45° C.

According to the present disclosure, the incubation period may be asshort as about one hour. In some embodiments, the first incubation isless than about 4 hours (e.g., less than about 2 hours, less than about3 hours, or less than about 4 hours. In some embodiments, the incubationperiod is less than about 8 hours (e.g., less than about 5 hours, lessthan about 6 hours, less than about 7 hours, or less than about 8hours). In some embodiments, the incubation is less than about 12 hours(e.g., about 9 hours, about 10 hours, about 11 hours, or about 12 hours.In some embodiments, the incubation period is less than or equal toabout 15 hours (e.g., less than about 13 hours, less than about 14hours, or less than about 15 hours. In some embodiments, the incubationperiod is up to 48 hours (e.g., less than about 24 hours, less thanabout 36 hours, or less than about 48 hours.

After the incubation period, the culture device is observed for anindication of the presence of an indicator microorganism. In someembodiments, the culture device is visually observed. In someembodiments, observing the culture device can comprise using an imagingdevice to observe the culture device. Imaging devices for scanning and,optionally, analyzing a culture device are known in the art and include,for example, PETRIFILM Plate Reader (PPR), available from 3M Company(St. Paul, Minn.), the PETRISCAN Colony Counter available from SpiralBiotech (Norwood, Mass.), and the PROTOCOL and ACOLYTE plate scannersavailable from Symbiosis (Cambridge, U.K.).

In the embodiments that utilize the optional second indicator system, anindication of the presence of an indicator microorganism can be observedby detecting a conversion of the second indicator system from a firststate to a second state. For example, in some embodiments, the secondindicator system may include a chromogenic reagent (e.g.,triphenyltetrazolium chloride or5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside) that exists in acolorless first state until it is converted (e.g., by indicatormicroorganisms) to a colored second state. In other embodiments, thesecond indicator system may include a fluorogenic reagent (e.g.,4-methylumbelliferyl β-D-galactoside or fluorescein diacetate) thatexists in a colorless first state until it is converted (e.g., byindicator microorganisms) to a fluorescent second state. In yet otherembodiments, the second indicator system may include a reagent (e.g., apH indicator) that can be converted from a first colored or fluorescentstate to a second colored or fluorescent state by a product of microbialactivity (e.g., the fermentation of a carbohydrate to acidend-products).

In some embodiments, detecting a conversion of the second indicatorsystem from a first state to a second state can comprise observing amicroorganism colony to detect the conversion. In some embodiments,detecting a conversion of the second indicator system from a first stateto a second state can comprise observing the culture medium to detectthe conversion.

FIG. 2 illustrates several aspects of detecting an indicatormicroorganism according to the present disclosure. FIG. 2 shows a topview of one embodiment of an inoculated culture device that includesindicator microorganisms reacting with a second indicator system. Theculture device has a culture medium 282 that includes a second indicatorsystem that includes a fermentable carbohydrate (e.g., glucose) and areagent (e.g., chlorophenol red) in a first state (the first state isillustrated as dark gray in FIG. 2; chlorophenol red exists in aviolet-colored first state in a culture medium having a pH about 6.8 orhigher). Also shown in FIG. 2 are indicator microorganism colonies 284that have fermented the glucose to diffusible acid end-products, whichreact with the reagent to convert it to a second state (the second stateis illustrated as a light gray halo 286 surrounding the colonies 284 inFIG. 2; chlorophenol red converts to a yellow-colored second state in aculture medium having a pH less than about 5.2). In some embodiments,the colonies may be stained by the reagent and may appear as the same(or a similar) color as the culture medium immediately surrounding them.In other embodiments (not shown), the second indicator system may simplycomprise an enzyme substrate or redox reagent that changes from a firststate to a second state and thereby directly changing the color orfluorescent properties of the colonies themselves, rather than theculture medium surrounding the colonies.

In the embodiments that do not utilize the optional second indicatorsystem (e.g., a culture device containing a selective agar culturemedium, not shown), an indication of the presence of an indicatormicroorganism can be detected by observing the presence of a bacterialcolony (e.g., by its typical size, shape, color, and/or morphology, asknown in the art) on or in the culture medium.

Table 1 shows several nonlimiting examples of selective culture mediathat can be used to support the growth of Enterobacteriaceaemicroorganisms, an exemplary group of indicator microorganisms thatindicate the presence of Salmonella target microorganisms. Also shown inTable 1 are exemplary indicator systems (i.e., second indicator systems,according to the present disclosure) that can be used to detect theEnterobacteriaceae microorganisms. Exemplary first indicator systemsthat can be used to indicate the presence of Salmonella targetmicroorganisms include caprylic acid esterase enzyme substrates(disclosed in PCT Patent Application Publication No. WO2007023185, whichis incorporated herein by reference in its entirety); 2-deoxy-D-riboseplus neutral red (disclosed in U.S. Pat. No. 7,150,977; which isincorporated herein by reference in its entirety);5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside (disclosed in U.S. Pat.No. 6,368,817; which is incorporated herein by reference in itsentirety); propanediol plus neutral red (disclosed in U.S. Pat. No.5,194,374; which is incorporated herein by reference in its entirety);and a combination of melibiose, mannitol, sorbitol, and neutral red(disclosed in U.S. Pat. No. 5,786,167; which is incorporated herein byreference in its entirety).

TABLE 1 Culture Medium Indicator System Violet Red Bile Agar Glucose +neutral red PETRIFILM Enterobacteriaceae Glucose + chlorophenol redCount Plate

An exemplary embodiment of a method of detecting a Salmonella targetmicroorganism according to the present disclosure includes using aculture device (e.g., an agar Petri plate or a PETRIFILM culture device)with a culture medium that includes the components listed in Table 2.When preparing the agar culture medium, all of the components except thesodium novobiocin and the cefsulodin are mixed together in any order andthereafter boiled and cooled to form a basal medium. The sodiumnovobiocin and the cefsulodin are added to the cooled, boiled basalmedium just prior to completion of the plating medium. In addition tothe culture medium, a detection article is prepared with a firstindicator system comprising5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside, as described inExample 1. The culture medium is inoculated with a sample and incubatedfor a period of time (e.g., about 18-48 hours). After incubation, theculture device is observed for an indication of the presence of at leastone indicator microorganism (e.g., a generally circular, red colonyand/or zone that indicates the fermentation of 2-deoxy-D-ribose to anacid end product that changes the neutral red pH indicator fromcolorless to red). The zone may have a visible colony in or near itscenter. If an indicator microorganism is detected, the detection articleis contacted with the culture medium and the article-contacted culturemedium can be incubated for a period of time (e.g., about 1-5 hours) andobserved for an indication of the presence of a target microorganism(i.e., a blue-colored colony generally in the center of a red zone thatwas formed from the reaction of the microorganisms with the firstindicator system. Optionally, an additional second indicator systemcomprising a chromogenic β-galactopyranoside substrate that produces adifferent color than the5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside) can be added to theculture medium or the detection article. The additional second indicatorsystem can be used to further identify non-target indicatormicroorganisms, such as certain strains of E. coli and Citrobacterfreundii. As an alternative to neutral red, other pH indicators known inthe art can be used in the second indicator system to detect acid endproducts from the fermentation of the 2-Deoxy-D-ribose. Examples ofother suitable pH indicators are disclosed herein. In some embodiments,other second indicator systems (e.g., triphenyltetrazolium chloride) canbe used in place of or in addition to the 2-Deoxy-D-ribose/neutral redindicator system to indicate the presence of indicator microorganisms.In some embodiments, other second indicators can be incorporated intothe detection article instead of the culture medium. Alternative firstindicator systems that could be included in the detection article mayinclude other chromogenic or fluorogenic enzyme substrates forα-galactosidase, chromogenic or fluorogenic enzyme substrates forcaprylate esterase, or indicator systems to detect phenylalaninedeaminase or propanediol fermentation.

TABLE 2 Component Grams/Liter Yeast Extract 3.00 Proteose Peptone 10.00Lab Lemco Powder 1.00 Sodium Chloride 5.00 L-phenylalanine 3.50 FerricAmmonium Citrate 0.50 Bile Salts #3 0.40 Bile Salts 0.202-deoxy-D-ribose 12.0 Neutral Red 0.03 Agar* 15.97 Deionized water* 950mL/liter Sodium novobiocin 0.02 Cefsulodin 0.006

Nonlimiting examples of selective culture media that can be used tosupport the growth of Enterobacteriaceae and/or coliform microorganisms,exemplary groups of indicator microorganisms that indicate the presenceof E. coli O157:H7 target microorganisms, include Violet RedBile-Glucose medium, Violet Red Bile-Lactose medium, PETRIFILM E. coliCount Plates, PETRIFILM Coliform Count plates, PETRIFILMEnterobacteriaceae Count plates, and PETRIFILM Rapid Coliform Countplates. Exemplary first indicator systems that can be used to indicatethe presence of E. coli O157:H7 target microorganisms include acombination of salicin, adonitol, inositol, and sorbitol with phenol red(disclosed in U.S. Pat. No. 6,617,149; which is incorporated herein byreference in its entirety).

Nonlimiting examples of selective culture media that can be used tosupport the growth of Listeria microorganisms, an exemplary group ofindicator microorganisms that indicate the presence of Listeriamonocytogenes target microorganisms, include Modified Oxfords Medium andR&F medium. Exemplary first indicator systems that can be used toindicate the presence of Listeria monocytogenes target microorganismsinclude phosphatidylinositol phospholipase C enzyme substrates(disclosed in U.S. Patent Application Publication No. 20070259393, whichis incorporated herein by reference in its entirety) and alphamannosidase enzyme substrates (disclosed in U.S. Pat. No. 7,351,548;which is incorporated herein by reference in its entirety). Non-limitingexamples of first indicator systems that can be used to detect Listeriamonocytogenes target microorganisms include5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate,5-Bromo-6-chloro-3-indoxyl myo-inositol-1-phosphate, ammonium salt,4-Methylumbelliferyl myo-inositol-1-phosphate, N-methyl-morpholine salt,3-indoxyl-α-D-mannopyranoside, 5-bromo-3-indoxyl-α-D-mannopyranoside,4-chloro-3-indoxyl-α-D-mannopyranoside,5-iodo-3-indoxyl-α-D-mannopyranoside,5-bromo-4-chloro-3-indoxyl-α-D-mannopyranoside,chloro-3-indoxyl-α-D-mannopyranoside,5-bromo-6-chloro-3-indoxyl-α-D-mannopyranoside,6-bromo-3-indoxyl-α-D-mannopyranoside,6-chloro-3-indoxyl-α-D-mannopyranoside,6-fluoro-3-indoxyl-α-D-mannopyranoside,4,6-dichloro-3-indoxyl-α-D-mannopyranoside,6,7-dichloro-3-indoxyl-α-D-mannopyranoside,4,6,7-trichloro-3-indoxyl-α-D-mannopyranoside,5-bromo-4-chloro-N-methyl-α-D-mannopyranoside,3-indoxyl-α-D-mannopyranoside, andN-methyl-3-indoxy-α-D-mannopyranoside,6-Bromo-2-naphthyl-α-D-mannopyranoside,4-Methylumbelliferyl-α-D-mannopyranoside, and4-Nitrophenyl-α-D-mannopyranoside.

In a preferred embodiment, a thin film culture device to detect thepresence of a Listeria microorganism can comprise a dehydrated brothmedium that includes proteose peptone, tryptone, casamino acids, LabLemco Powder/beef extract, glucose, yeast extract, dipotassium hydrogenphosphate, lithium chloride, bovine serum albumin, guar gum, nalidixicacid (sodium salt), chromogenic enzyme substrates to detectb-glucosidase enzyme activity (e.g., Salmon-β-D-glucoside and/orMagenta-β-D glucoside), and ceftazidime pentahydrate. A liquid sample isused to inoculate the medium and a Listeria microorganism, if present,grows and forms colonies that are red-colored due to the hydrolysis ofthe β-glucosidase chromogenic enzyme substrates. If a Listeriamicroorganism is detected in the culture device, the inoculated mediumin the culture device optionally can be contacted with a detectionarticle comprising a dehydrated coating that includes a chromogenicindicator to detect phosphatidylinositol phospholipase Cenzyme activity(e.g., 5-Bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate), guar gumand a phosphate buffer. If one or more of the colonies include Listeriamonocytogenes microorganisms, it will hydrolyse the phosphatidylinositolphospholipase C enzyme substrate, causing the colonies to turn blue.Advantageously, the detection article need only be used if a Listeriamicroorganism is first detected by observing evidence of β-glucosidaseenzyme activity associated with a colony.

Nonlimiting examples of selective culture media that can be used tosupport the growth of Enterobacteriaceae microorganisms, an exemplaryindicator microorganism that indicates the presence of Cronobactersakazakii target microorganisms, include Violet Red Bile-Glucose mediumand PETRIFILM Enterobacteriaceae Count plates. Exemplary first indicatorsystems that can be used to indicate the presence of Cronobactersakazakii target microorganisms include chromogenic alpha glucosidaseand beta cellobiosidase enzyme substrates (disclosed in U.S. PatentApplication Publication No. 2006/0257967 which is incorporated herein byreference in its entirety).

In some embodiments, a group of indicator microorganisms that is knownto be found in a similar environment (e.g., fecal material) as thetarget microorganism may not actually include the target microorganism.An example of this condition is illustrated by the relationship ofcoliform indicator microorganisms and Salmonella target microorganisms.Coliform bacteria are found in fecal material and are characterized bythe ability to ferment lactose to acid end products. Certain Salmonellatarget microorganisms (e.g., Salmonella enterica Typhimurium) are foundin fecal material and do not ferment lactose to acid end products. Thus,a method according to the present disclosure may include providing afirst indicator system (e.g.,5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside) to detect Salmonellatarget microorganisms and a second indicator system (e.g., lactose andneutral red) to detect coliform indicator microorganisms. In thisembodiment, colonies reacting with the first indicator system would notreact with the second indicator system.

The observation of an indication of the presence of an indicatororganism connotes the possible presence of a target microorganism (e.g.,a potential pathogen) in the sample. Thus, it is a feature of the methodthat, when an indication of the presence of indicator microorganisms isobserved, a detection article can be used to confirm the presence orabsence of the target microorganism in the sample. Conversely, in someembodiments where an indication of the presence of indicatormicroorganisms is not observed in the culture device, the use of adetection article in the method can be avoided because the absence of anindicator microorganism in the sample connotes the absence of a targetmicroorganism in the sample.

Methods of the present disclosure comprise providing a detection articlecomprising a first indicator system. FIG. 3 shows a perspective view ofone embodiment of a detection article 300 according to the presentdisclosure. The detection article 300 comprises a solid support 310 withupper and lower major surfaces and a coating 320 disposed thereon. Thecoating 320 comprises a first indicator system, as described herein. Thefirst indicator system can be selected to detect a particular targetmicroorganism. Optionally, an adhesive layer (not shown) may be disposedon the solid support 310 between the solid support 310 and the coating320. The optional adhesive layer should comprise an adhesive that doesnot substantially interfere with the reaction of the first indicatorsystem with the target microorganism and/or interfere with theobservation of the second indicator system. A nonlimiting example of asuitable adhesive includes the isooctylacrylate/acrylamide (94:6)pressure-sensitive adhesive described in U.S. Pat. No. 4,565,783; whichis incorporated herein by reference in its entirety.

In some embodiments, the coating 320 is substantially water-free (i.e.,it has a water content no greater than about the water content of thedehydrated coating once it has been allowed to equilibrate with theambient environment). In some embodiments, the coating 320 can beapplied to the substrate 310 or the optional adhesive layer (not shown)as a dry coating such as a powder or the coating can be applied to thesubstrate as a liquid that is subsequently dried on the substrate, bothprocesses as described, for example, in U.S. Pat. No. 4,565,783. In someembodiments, the article 300 can be constructed, coated, and dried asdescribed for the manufacture of the composite in U.S. Pat. No.6,022,682, which is incorporated herein by reference in its entirety.

In some embodiments, the coating 320 comprises a binder. There are manybinders that would be suitable for use in the detection article 300.Nonlimiting examples of suitable binders include agarose, guar gum,xanthan gum, locust bean gum, and other natural gums. A preferred binderis guar gum.

The coating 320 preferably may also include other constituents such as,for example, a suitable buffering agent to control the pH at a pointwhere a reaction between the first indicator system and the targetmicroorganisms is facilitated. The choice of the particular bufferingagent (e.g. a phosphate buffer) and pH (e.g., 7.2) may depend upon thefirst indicator system and/or the target microorganism, as will berecognized by a person having ordinary skill in the art.

The solid support 310 should be selected from materials that can becoated and that do not substantially obscure the observation of thereactants or products of the first indicator system and, optionally, thesecond indicator system. The solid support 310 may be a polymer film,such as a polyester film. The solid support 310 may be derived from asheet material (e.g., polymer film, paper, nonwoven), allowing forcutting, or punching, of detection articles 300 of desired size or shapefollowing coating and drying. In some embodiments, the solid support 310may be transparent or translucent or it may become transparent ortranslucent when placed in contact with a hydrogel. The material usedfor the solid support 310 may be selected to impart any degree ofrigidity or flexibility to the detection article 300. In addition, thedetection article 300 can be prepared in any shape (e.g., circular,ovoid, square, rectangular, etc.) or thickness, depending on what isdesired for a particular application.

The solid support 310 preferably is transparent or at least translucent,to allow the viewing of color changes that develop when the article isplaced in fluidic contact with a culture device. The solid support 310also provides stability to the article and protects it from damage.

The solid support 310 may be selected such that it is peelable from thecoating 320, leaving the coating free for use in testing (e.g., influidic contact with a culture medium) without the solid support 310.For example, where a polyester film is used as the solid support, thesolid support 310 may be peelable from the coating 320 when the coating320 becomes hydrated after contact with the culture device.

In use, the detection article is brought into fluidic contact with theculture medium of the culture device. Preferably, the detection articleand the culture medium will be brought into contact in the culturedevice (e.g., by placing the detection article in contact with theculture medium in the culture device). In certain preferred embodiments,the detection article is dimensioned so that it has substantially thesame shape and surface area so that the article contacts an entiresurface of the culture medium in the culture device. In someembodiments, the detection article may be prehydrated (e.g., withsterile water or buffer), although it is contemplated that the moisturein the culture medium is sufficient to hydrate the detection article,thereby permitting the first indicator system to come into fluidiccontact with microorganisms present in the culture device.

Without being bound by theory, it is believed that the fluidic contactbetween the culture medium and the detection article permits thediffusion of the first indicator system, a metabolite produced by themicroorganism, and/or the microorganism (or component thereof, such asan enzyme, for example) such that a component of the first indicatorsystem can react with the metabolite, the target microorganism, or acomponent of the target microorganism and be converted from a firststate to a second state. Thus, in some embodiments, the conversion ofthe first indicator system from a first state to a second state, ifpresent, is indicative of the presence of at least one targetmicroorganism.

In some embodiments, the conversion of the first indicator system from afirst state to a second state, if absent, is indicative of the presenceof at least one target microorganism. In these embodiments of themethod, when an indicator microorganisms changes the first indicatorsystem from a first state to a second state (e.g., the colony and/or theculture medium immediately surrounding the colony becomes colored orfluorescent), then the indicator microorganism is not the targetmicroorganism. Conversely, in these embodiments, if the indicatormicroorganism does not change the first indicator system from a firststate to a second state, it is an indication that the indicatormicroorganism is the target microorganism. A particular nonlimitingexample can illustrate this point. Certain microorganisms of the genusShigella are pathogenic to humans and, thus, Shigella is an example of atarget microorganism. Shigella is a genus of the Enterobacteriaceaefamily. Because Enterobacteriaceae microorganisms (including Shigellaspecies) can be found in fecal material, Enterobacteriaceaemicroorganisms are an example of indicator microorganisms for Shigella.In an embodiment of the method of the present disclosure, a sample ofmaterial can be inoculated on selective culture media forEnterobacteriaceae (e.g., violet red bile agar or PETRIFILMEnterobacteriaceae Count Plates). If indicator microorganisms aredetected on the culture medium, a detection article comprising, forexample, a chromogenic enzyme substrate to detect β-glucosidase,β-fucosidase, and/or N-acetyl-β-galactosaminidase can contacted with theculture medium. A conversion of any one of those enzyme substrates froman uncolored state to a colored state is an indication that the colonyis not the target microorganism because Shigella target microorganismsdo not comprise any of the corresponding enzyme activities for thoseenzyme substrates.

After bringing the detection article into fluidic contact with theculture medium, the article-contacted culture medium is observed todetect a conversion of the first indicator system from a first state toa second state (e.g., from a colorless state to a colored state, from anonfluorescent state to a fluorescent state, from a first color to asecond color). This conversion can be detected, for example, by any ofthe detection means discussed herein to detect a conversion of thesecond indicator system from a first state to a second state.Optionally, in some embodiments, the article-contacted culture mediumcan be incubated at a predetermined temperature for a period of time tofacilitate the conversion of the first indicator system from a firststate to a second state. The incubation temperature can be, for example,ambient temperature (about 23° C.), up to about 28° C., up to about 30°C., up to about 35° C., up to about 37° C., up to about 42° C., or up toabout 45° C. In some embodiments, the article-contacted culture mediumcan be incubated at the same temperature as the incubation temperatureused to grow and detect the indicator microorganisms. In someembodiments, the article-contacted culture medium can be incubated at adifferent (e.g., lower or higher) temperature than the incubationtemperature used to grow and detect the indicator microorganisms.

In some embodiments, the article-contacted culture medium can beincubated for at least about 15 minutes. In some embodiments, thearticle-contacted culture medium can be incubated for at least about 30minutes. In some embodiments, the article-contacted culture medium canbe incubated for at least about 60 minutes. In some embodiments, thearticle-contacted culture medium can be incubated for at least about 90minutes. In some embodiments, the article-contacted culture medium canbe incubated for at least about 2 hours. In some embodiments, thearticle-contacted culture medium can be incubated for at least about 4hours. In some embodiments, the article-contacted culture medium can beincubated up to about 60 minutes. In some embodiments, thearticle-contacted culture medium can be incubated up to about 90minutes. In some embodiments, the article-contacted culture medium canbe incubated up to about 2 hours. In some embodiments, thearticle-contacted culture medium can be incubated up to about 3 hours.In some embodiments, the article-contacted culture medium can beincubated up to about 4 hours. In some embodiments, thearticle-contacted culture medium can be incubated up to 5 hours. In someembodiments, the article-contacted culture medium can be incubated up to8 hours. In some embodiments, the article-contacted culture medium canbe incubated up to 12 hours. In some embodiments, the article-contactedculture medium can be incubated up to 24 hours. In some embodiments, thearticle-contacted culture medium can be incubated between about 15minutes and about 8 hours, inclusive. In some embodiments, thearticle-contacted culture medium can be incubated between about 30minutes and about 8 hours, inclusive. In some embodiments, thearticle-contacted culture medium can be incubated between about 30minutes and about 5 hours, inclusive. In some embodiments, thearticle-contacted culture medium can be incubated between about 1 hourand about 5 hours, inclusive. In some embodiments, the article-contactedculture medium can be incubated between about 2 hours and about 5 hours,inclusive. In some embodiments, the article-contacted culture medium canbe incubated between about 2 hours and about 4 hours, inclusive.

FIG. 4 shows a top view of the culture device of FIG. 3 after placementof a detection article in contact with the culture (growth) medium inthe device. The culture device has a culture medium 482 on and/or inwhich colonies grow. As shown in FIG. 2, indicator microorganismcolonies 484 are present and have changed the first indicator systemfrom a first state (dark gray) to a second state (light gray), forming adistinctive halo 486 around the indicator microorganism colonies 484.Also shown in FIG. 4 are target microorganism colonies 485, which appearlarger than the indicator microorganism colonies 484. This can be due,for example, to the conversion of the second indicator system from afirst state to a second state. In some embodiments, the second indicatorsystem may be a precipitable chromogenic enzyme substrate (e.g.,5-bromo-4-chloro-3-indolyl-β-D-glucuronide) which, when reacted with atarget microorganism, can turn the colony blue and may make the colonyappear slightly larger. The target microorganism colonies 485 also havereacted with the first indicator system and have halos 486 surroundingthem.

Methods of the present disclosure optionally can further comprise thestep of enumerating a type of microorganism. Enumerating a type ofmicroorganism comprises counting a number of colonies (or colony-formingunits) of the particular type of microorganism. The number of enumeratedcolony-forming units can be used to estimate the number ofmicroorganisms per gram (or per milliliter) in the original sample. Thenumber of enumerated colony-forming units can be compared to aspecification to determine whether the original sample complies with aquality standard, for example. In some embodiments, all of themicroorganisms of a particular type (e.g., indicator microorganisms,target microorganisms) can be enumerated. In some embodiments, a portion(e.g. up to a predetermined threshold number) of the microorganisms of aparticular type can be enumerated.

In some embodiments, enumerating a type of microorganisms comprisesenumerating the number of indicator microorganisms. In some embodiments,enumerating a type of microorganisms comprises enumerating the number oftarget microorganisms. In some embodiments, enumerating a type ofmicroorganisms comprises enumerating the number of indicatormicroorganisms and the number of target microorganisms. In someembodiments, enumerating a type of microorganism can comprise using animaging device to enumerate the microorganisms.

EMBODIMENTS

Embodiment 1 is a method of detecting the presence or absence of atarget microorganism, comprising:

providing:

-   -   a culture device including a culture medium comprising        ingredients selected to facilitate growth of a predetermined        indicator microorganism;    -   a detection article comprising a first indicator system, the        first indicator system selected to detect a target        microorganism; and    -   a sample;

inoculating the culture device with the sample;

incubating the inoculated culture device for a period of time sufficientto permit growth of the indicator microorganisms;

observing the culture device for an indication of a presence of at leastone indicator microorganism;

contacting the culture medium of the incubated culture device with thedetection article; and

observing the article-contacted culture device to detect a conversion ofthe first indicator system from a first state to a second state.

Embodiment 2 is method of embodiment 1 wherein the conversion of thefirst indicator system from a first state to a second state, if present,is indicative of the presence of at least one target microorganism.

Embodiment 3 is the method of embodiment 1, wherein the conversion ofthe first indicator system from a first state to a second state, ifabsent, is indicative of the presence of at least one targetmicroorganism.

Embodiment 4 is the method of any one of the preceding embodiments,further comprising providing a second indicator system and placing thesecond indicator system in fluid communication with the culture medium,wherein observing the culture device for an indication of the presenceof at least one indicator microorganism comprises detecting a conversionof the second indicator system from a first state to a second state.

Embodiment 5 is the method of any one of the preceding embodiments,wherein providing the culture device further comprises providing aculture device that comprises a hydrogel or a dry, cold-water-solublegelling agent.

Embodiment 6 is the method of any one of the preceding embodiments,wherein contacting the culture medium with the detection article isperformed only when the indication of the presence of at least oneindicator microorganism is observed.

Embodiment 7 is the method of any one of the preceding embodiments,wherein providing a culture device further comprises providing a culturedevice that includes the second indicator system.

Embodiment 8 is the method of any one of embodiments 1 through 7,wherein providing a culture medium comprises providing a culture mediumselected to facilitate growth of an Enterobacteriaceae microorganism,wherein providing a detection article comprises providing a detectionarticle to detect a microorganism of the genus Salmonella.

Embodiment 9 is the method of embodiment 8, wherein the first indicatorsystem comprises a reagent to detect α-galactosidase or caprylateesterase enzyme activity.

Embodiment 10 is the method of any one of embodiments 1 through 7,wherein providing a culture medium comprises providing a culture mediumselected to facilitate growth of an Enterobacteriaceae microorganism,wherein providing a detection article comprises providing a detectionarticle to detect a microorganism of the genus Shigella.

Embodiment 11 is the method of embodiment 10, wherein the firstindicator system comprises a reagent to detect β-glucosidase,β-fucosidase, N-acetyl-β-galactosaminidase, or a combination of any twoor more of the foregoing enzyme activities.

Embodiment 12 is the method of any one of embodiments 1 through 7,wherein providing a culture medium comprises providing a culture mediumselected to facilitate growth of an Enterobacteriaceae microorganism,wherein providing a detection article comprises providing a detectionarticle to detect a microorganism of the genus Cronobacter.

Embodiment 13 is the method of embodiment 12, wherein the firstindicator system comprises a reagent to detect α-glucosidase and/orβ-cellobiosidase enzyme activity.

Embodiment 14 is the method of any one of embodiments 1 through 7,wherein providing a culture medium comprises providing a culture mediumselected to facilitate growth of an Enterobacteriaceae microorganism,wherein providing a detection article comprises providing a detectionarticle to detect Escherichia coli.

Embodiment 15 is the method of embodiment 14, wherein the firstindicator system comprises a reagent to detect β-glucuronidase enzymeactivity.

Embodiment 16 is the method of any one of embodiments 1 through 7,wherein providing a culture medium comprises providing a culture mediumselected to facilitate growth of a coliform microorganism, whereinproviding a detection article comprises providing a detection article todetect Escherichia coli.

Embodiment 17 is the method of embodiment 16, wherein the firstindicator system comprises a reagent to detect β-glucuronidase enzymeactivity.

Embodiment 18 is the method of any one of embodiments 1 through 7,wherein providing a culture medium comprises providing a culture mediumselected to facilitate growth of a microorganisms of the Listeria genus,wherein providing a detection article comprises providing a detectionarticle to detect Listeria monocytogenes.

Embodiment 19 is the method of embodiment 18, wherein the firstindicator system comprises a reagent to detect α-mannopyranosidaseand/or phosphatidylinositol-specific phospholipase C enzyme activity.

Embodiment 20 is the method of any one of embodiments 1 through 19,wherein observing the culture device or the article-contacted culturedevice comprises observing the culture device visually.

Embodiment 21 is the method of any one of embodiments 1 through 19,wherein observing the culture device or the article-contacted culturedevice comprises observing the culture device using an imaging device.

Embodiment 22 is the method of any one of the preceding embodiments,further comprising enumerating indicator microorganism colony-formingunits in the culture device.

Embodiment 23 is the method of any one of the preceding embodiments,further comprising enumerating target microorganism colony-forming unitsin the culture device.

Embodiment 24 is the method of any one of the preceding embodiments,wherein contacting the culture medium with the detection article furthercomprises contacting the culture medium with the detection article at apredetermined temperature.

Embodiment 25 is a detection article comprising a substrate with upperand lower major surfaces and a coating comprising a first indicatorsystem disposed on at least one of the major surfaces, wherein the firstindicator system is converted from a first state to a second state byα-galactopyranoside or caprylate esterase enzyme activity.

Embodiment 26 is the article of embodiment 25, wherein the firstindicator system includes an indicator selected from the groupconsisting of 5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside,5-bromo-6-chloro-3-indolyl-caprylic acid,5-bromo-4-chloro-3-indolyl-caprylic acid, and a combination of any twoor more of the foregoing indicators.

Embodiment 27 is a detection article comprising a substrate with upperand lower major surfaces and a first indicator system coated on at leastone of the major surfaces, wherein the first indicator system isconverted from a first state to a second state by β-glucuronidase enzymeactivity.

Embodiment 28 is the article of embodiment 27, wherein the firstindicator system includes an indicator selected from the groupconsisting of 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid,p-nitrophenyl-β-glucuronide,p-nitrophenyl-2,3,4-tri-O-acetyl-β-glucuronic acid methyl ester,phenolphthalein glucuronic acid, phenolphthalein mono-P-glucuronic acid,naphthyl-AS-BI-β-D-glucuronide, 4-methylumbelliferyl β-D-glucuronide,8-Hydroxyquinoline-beta-D-glucuronic acid, sodium salt,2-Naphthyl-beta-D-glucuronic acid, sodium salt,4-Nitrophenyl-beta-D-glucuronic acid, sodium salt,Phenolphthalein-beta-D-glucuronic acid, sodium salt monohydrate,5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, cyclohexylammoniumsalt, 3-Indoxyl-beta-D-glucuronic acid, cyclohexylammonium salt,3-Indoxyl-beta-D-glucuronic acid, sodium salt,5-Bromo-6-chloro-3-indoxyl-beta-D-glucuronic acid, cyclohexylammoniumsalt, 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, sodium saltanhydrous, and 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, sodiumsalt trihydrate, and a combination of any two or more of the foregoingindicators.

Embodiment 29 is a detection article comprising a substrate with upperand lower major surfaces and a first indicator system coated on at leastone of the major surfaces, wherein the first indicator system isconverted from a first state to a second state by α-mannopyranosidase orphosphatidylinositol-specific phospholipase C enzyme activity s.

Embodiment 30 is the article of embodiment 29, wherein the firstindicator system includes an indicator selected from the groupconsisting of 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate,5-Bromo-6-chloro-3-indoxyl myo-inositol-1-phosphate, ammonium salt,4-Methylumbelliferyl myo-inositol-1-phosphate, N-methyl-morpholine salt,3-indoxyl-α-D-mannopyranoside, 5-bromo-3-indoxyl-α-D-mannopyranoside,4-chloro-3-indoxyl-α-D-mannopyranoside,5-iodo-3-indoxyl-α-D-mannopyranoside,5-bromo-4-chloro-3-indoxyl-α-D-mannopyranoside,chloro-3-indoxyl-α-D-mannopyranoside,5-bromo-6-chloro-3-indoxyl-α-D-mannopyranoside,6-bromo-3-indoxyl-α-D-mannopyranoside,6-chloro-3-indoxyl-α-D-mannopyranoside,6-fluoro-3-indoxyl-α-D-mannopyranoside,4,6-dichloro-3-indoxyl-α-D-mannopyranoside,6,7-dichloro-3-indoxyl-α-D-mannopyranoside,4,6,7-trichloro-3-indoxyl-α-D-mannopyranoside,5-bromo-4-chloro-N-methyl-α-D-mannopyranoside,3-indoxyl-α-D-mannopyranoside, andN-methyl-3-indoxy-α-D-mannopyranoside,6-Bromo-2-naphthyl-α-D-mannopyranoside,4-Methylumbelliferyl-α-D-mannopyranoside,4-Nitrophenyl-α-D-mannopyranoside and a combination of any two or moreof the foregoing indicators.

Embodiment 31 is the article of any one of embodiments 25 through 30,further comprising an adhesive layer, wherein at least a portion of thefirst indicator system is disposed on or in the adhesive layer.

Embodiment 32 is the article of any one of embodiments 25 through 31,wherein the first indicator system is coated on both major surfaces.

Embodiment 33 is the article of any one of embodiments 25 through 32,wherein the substrate is selected from the group consisting of apolymeric film, paper, a nonwoven, a membrane filter and derivatives ofany of the foregoing.

Embodiment 34 is the article of any one of embodiments 25 through 33,wherein the coating comprises a binder.

EXAMPLES

Materials

-   Potassium Phosphate Monobasic (KH₂PO₄)—Mallinckrodt Baker, Inc.;    Phillipsburg, N.J.-   Potassium Phosphate Dibasic (K₂HPO₄)—AMRESCO; Solon, Ohio-   Guar gum—M150 guar MEYPROGAT gum, Meyhall Chemical AG-   5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside—BIOSYNTH AG,    Rietlistr, Switzerland-   Polyester film—2.91 mil (0.074 mm) clear polyester film-   BCIG—5-Bromo-4-chloro-3 Indolyl-beta-D-glucuronide acid    Cyclohexylammonium Salt—BIOSYNTH AG-   Methyl Glucuronide—1-O-Methyl-beta-D-glucuronic Acid, Sodium    Salt—BIOSYNTH AG

Example 1. Preparation of a Detection Article to Detect a SalmonellaTarget Microorganism

A coating composition was prepared by adding 13.6 g of potassiumphosphate monobasic in 1000 mL of reverse osmosis treated water in a 4 Lcontainer and mixing with an air mixer for about 1.5 minutes followed byadding 3.4 g of potassium phosphate dibasic and mixing about 2 minutes.Then 11 g of guar gum was slowly added and mixed for about 5 minutes.The container was covered and heated on a hot plate, while mixing, untilthe mixture reached 80° C. The container was removed from the hot plateand mixed at room temperature for about 15 minutes, and thenrefrigerated until the mixture was about 40° C. An indicator suspensionwas prepared by adding the5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside to 10 mL of reverseosmosis water and vortexing the mixture to form a uniform suspension.After cooling the coating composition to about 40° C., the indicatorsuspension was mixed into the coating composition.

The mixture was then removed from the refrigerator and allowed to cometo approximately room temperature. The mixture was manually knife coatedto a width of about 8 inches (20.32 cm) on a 10 inch (25.4 cm) by 54inch (137.2 cm) sheet of polyester film to obtain a dry coating weightof about 0.150 grams per 24 square inches (9.69 g/m², “Disk 1A”). Thecoating procedure was repeated to provide coated films having drycoating weights of 0.185 g/24 square inches (11.95 g/m², “Disk 1B”),0.240 g/24 square inches (15.5 g/m², “Disk 1C”), and 0.30 g/24 squareinches (19.38 g/m², “Disk 1D”). The coated sheets were dried at an ovenset at 230F (110 C) for 5 to 15 minutes until dry. The coated sheetswere stored in plastic bags. The sheets were each cut into 4 inch (10.2cm) squares and the squares were kept in plastic bags until tested.

Example 2. Detecting a Salmonella Target Microorganism with a DetectionArticle

A colony of Salmonella enterica Agona ((FSD #140) isolated from an agarstreak plate, was inoculated into 5 mL of tryptic soy broth andincubated overnight at 35° C. The overnight culture was diluted inButterfield's Phosphate Diluent to obtain a suspension havingapproximately 100 colony-forming units (CFU) per milliliter. Fourbacterial culture devices (3M™ Petrifilm™ Enterobacteriaceae CountPlates; 3M Company; St. Paul, Minn.) were each inoculated with 1 mL ofthe bacterial suspension according to the manufacturer's instructions,and incubated at 35° C. for 22-24 hours. The devices were inspected andred colonies (about 1 mm diameter) surrounded by yellow acid zones(approximately 24 mm in diameter) The top film of the culture plate wascarefully lifted away from the bottom film to expose the culture mediumin the culture plate. Two square disks (Disk1A) were inserted into theplate. The first disk was placed with its coated surface against theculture medium adhered to the bottom film (i.e., facing downward). Thesecond disk was placed on top of the first disk with the second disk'scoated side facing away from the bottom film (i.e., facing upward). Thetop film was carefully lowered (using a “rolling” motion) to bring theculture medium adhered to the top film into contact with the coated sideof the second disk. Light finger pressure was applied to the outersurface of the top film of the closed plate to ensure contact betweenthe surfaces of the culture device and the coated surfaces of the disks.The procedure was repeated with Disks 1B, 1C, and 1D. The devices withthe disks were incubated at 35° C. and inspected every hour for fivehours. Digital images were taken during each inspection. The imagesshowed that, within about 3 hours, all of the colonies on each of theplates turned blue to blue-green due to the hydrolysis of the5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside by the microorganismsin the colonies.

Example 3. Preparation of a Detection Article to Detect an E. ColiTarget Microorganism

A coating composition was prepared according to the procedure ofExample 1. A suspension was prepared by dissolving 0.4 g of1-O-methyl-β-D-glucuronic acid (sodium salt) and 2.0 grams of5-bromo-4-chloro-3-indoxyl-β-D-glucuronic acid in 50 mL of reverseosmosis treated water in a beaker with constant stirring at roomtemperatures with a magnetic stir bar. The beaker containing indicatorsuspension was placed in a refrigerator.

The coating composition was removed from the refrigerator when it hadcooled to approximately 40° C. and subsequently was mixed with an airmotor mixer until a vortex formed. The indicator suspension was added tothe coating composition and mixed for about 20 minutes. The resultingdetection medium was then covered and refrigerated until used.

Disks 2A, 2B, 2C, and 2D were prepared and stored according to theprocedure described in Example 1 having dry coating weights of 0.150grams per 24 square inches (9.69 g/m², “Disk2A”), 0.185 g/24 squareinches (11.95 g/m², “Disk2B”), 0.240 g/24 square inches (15.5 g/m²,“Disk2C”), and 0.30 g/24 square inches (19.38 g/m², “Disk2D”).

Example 4. Detecting an E. Coli Target Microorganism with a DetectionArticle

An isolated colony of E. coli strain (ATCC #51813) was inoculated into 5mL of tryptic soy broth and incubated 35° C. for 20 hours to provide anovernight culture having a bacterial concentration of approximately2×10⁹ cfu/mL. The culture was vortexed and 10 microliters of the culturewas added to 99 mL of Butterfields Phosphate Buffer (Dilution1). Thesuspension was shaken vigorously for about 20 seconds, and 5 microlitersof this dilution was added to another 99 mL of Butterfields PhosphateBuffer (Dilution2). This suspension was shaken vigorously for about 20seconds. Sixteen bacterial culture devices (3M™ Petrifilm™High-Sensitivity Coliform Count Plate; 3M Company; St. Paul, Minn.) wereeach inoculated with 5 mL of Dilution 2 according to the manufacturer'sinstructions. Four culture devices were incubated at each of 4temperatures (32° C., 35° C., 37° C., and 44.5° C., respectively) for22-24 hours. The culture devices were removed from the incubators and adigital image of each plate was recorded with a digital camera. Eachcolony had a characteristic appearance (i.e., red colony center about 1mm in diameter surrounded by a red zone about 2-3 mm in diameter) of acoliform colony on the High-Sensitivity Coliform Count Plate.

Disks of each type (i.e., Disk2A, Disk 2B, Disk 2C, and Disk 2D) wereplaced as described in Example 2 into separate culture device that hadbeen incubated at each temperature The devices with the disks were thenincubated at their respective temperature for 5 hours. A digital imageof each culture device was recorded after each hour of the 5-hourincubation period.

After the culture devices containing the disks were incubated, they wereobserved. Each of the red colonies on all of the plates turned blueafter the culture device was contacted with the disk and incubated for 5hours. Plates that were incubated at a higher temperature with the diskswere observed to have blue colonies at an earlier time than the platesthat were incubated at lower temperatures. Plates that receiveddetection articles with higher coat weights also were observed to haveblue colonies at an earlier time than the plates that received detectionarticles with lower coat weights.

Example 5. Prophetic Example of the Preparation of a Culture Device forGrowing Listeria Indicator Microorganisms

A culture medium composition is prepared with the materials in Table 3.The Proteose Peptone, Tryptone, Casamino acids, and Yeast Extract can beobtained, for example, from Becton Dickinson (Sparks, Md.). The LabLemco Powder/Beef Extract can be obtained, for example, from Oxoid(Hampshire, UK). Bovine serum albumin can be obtained from, for example,from Serologicals Corporation (Norcross, Ga.). Beta-D-glucosideindicators can be obtained from BioSynth AG, for example. Ceftazidimecan be obtained from Glaxo Wellcome, for example.

TABLE 3 Component Material Grams  1 Proteose peptone 6.0  2 Tryptone19.2  3 Casamino acids 12.0  4 Lab Lemco Powder/Beef Extract 10.0  5Glucose 5.0  6 Yeast extract 14.0  7 Potassium phosphate, dibasic 9.0  8Lithium chloride 15.0  9 Bovine serum albumin 6.0 10 Guar gum 10 11Nalidixic acid, sodium salt 0.02 12 Salmon-beta-D glucoside 0.06 13Magenta-beta-D glucoside 0.066 14 Ceftazidime pentahydrate 0.027

A first mixture is prepared by adding components 1-10 (shown in Table 3)to 1000 mL of reverse-osmosis water, mixing to obtain a uniformsuspension, heating the mixture until it reaches 80° C. and then coolingto room temperature. While the first mixture is cooling, a secondmixture is prepared by mixing the components 11-14 (shown in Table 1 in10 milliliters of reverse osmosis water. The second mixture is vortexedto provide a uniform mixture, which is added to the first mixture, andthen mixed further to provide a uniform culture medium. The culturemedium is then refrigerated for 18-24 h (2-8° C.).

The refrigerated culture medium is allowed to warm to room temperatureand then knife coated onto a sheet of 2.6 mil (0.074 mm) thick clearpolyester film and dried at about 200° F. for 5 to 10 minutes. The knifegap is adjusted to provide a dry coating weight of approximately 515-540mg/24 in² (33.27-34.88 g/m²). An 18 mil (0.46 mm) thick sheet of closedcell polystyrene foam is laminated to a pressure-sensitive adhesivetransfer tape. Circular openings 2 inches in diameter are die punchedfrom the foam sheet and the foam sheet is laminated to the culturemedium-coated side of the polyester film to form a structure similar tothe bottom portion of the culture device shown in FIG. 1 of U.S. Pat.No. 4,565,783. The preparation of the culture device is completed byremoving the top film from a 3M™ Petrifilm™ Staph Express Count Plateand affixing it to the Styrofoam sheet with double coated adhesive tape,thereby producing a device that resembles the complete culture deviceshown in FIG. 1 of U.S. Pat. No. 4,565,783.

Example 6. Prophetic Example of the Preparation of a Detection Articleto Detect a Listeria Monocytogenes Target Microorganism

A coating composition is made by mixing the guar gum, disodiumphosphate, and potassium hydrogen phosphate (shown in Table 4) in 1000milliliters of reverse osmosis water to form a uniform dispersion asdescribed in Example 1.

TABLE 4 Material Grams 5-Bromo-4-chloro-3-indoxyl-myo-inositol-1- 0.60phosphate Guar gum 10 Sodium phosphate, dibasic 8.0 Potassium phosphate,monobasic 4.0

The container with the coating composition is covered and the dispersionis mixed continuously while heated to a temperature of 80° C. Then it isremoved from the heat and mixed continuously at room temperature. Anindicator suspension is prepared by adding the5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate (which can beobtained from Biosynth AG, for example) to 10 mL of reverse osmosiswater and vortexing to form a uniform suspension. After the coatingcomposition cools to room temperature, the indicator suspension is mixedinto the coating composition. The mixture is covered and refrigerated18-22 hours. The coating mixture is removed from the refrigerator andallowed to warm to about room temperature. A 2.9 mil (0.074 mm)polyester film is knife coated with the mixture according to theprocedure described in Example 1 to obtain a film with a dry coatingweight of about 80-95 mg/24 in² (5.17-6.14 g/m²). The polyester film isturned over and knife-coated on the other side in the same manner. Thefilm, coated on both sides is then die-cut into circular disks having adiameter of about 2 inches.

Example 7. Prophetic Example of Detecting a Listeria MonocytogenesTarget Microorganism

Enrichment cultures are prepared from food and environmental samplesusing standard sample-collecting and enrichment broth procedures, (e.g.,as specified by the US Department of Agriculture, the Food SafetyInspection Service, or the Bacteriological Analytical Manual). Eightserial, 10-fold dilutions of the culture are prepared in Butterfield'sdiluent. One milliliter of each dilution is inoculated into individualculture devices described in Example 5. The devices are then incubated35-37° C. for 18-30 hours. The devices are inspected for typical colonymorphology (e.g., red colonies approximately 0.5 to 1.5 mm in diameter)indicative of Listeria (an indicator microorganism group that indicatesthe presence of Listeria monocytogenes). If typically colored coloniesare seen in the culture device after incubation, the user can furtherinsert a detection article to detect differentiate the colonies. The topfilm of the culture device is gently pulled back and a 2 inch detectionarticle described in Example 6 is placed into contact with the culturemedium. The top film is re-closed, bringing it into contact with thedetection article. The top film can be pressed with light fingerpressure to contact the growth areas of the device with the detectionarticle. The device is incubated for up to 5 hours at 35-37° C. andinspected periodically (e.g., hourly) for the presence of blue toblue-green color in the colonies. The blue to blue-green-coloredcolonies indicate the presence of L. monocytogenes.

Example 8. Method of Detecting a Listeria Monocytogenes TargetMicroorganism

Preparation of Thin Film Culture Device—Broth-Coated Substrate:

All of the ingredients shown in Table 5 except the guar gum were mixedwith 970 milliliters of deionized water in stainless steel beaker. Whilemixing, the guar was added and the complete mixture was heated to 80° C.The heated complete mixture was stirred for about 15 minutes, covered,and cooled in a refrigerator overnight. Prior to coating, twentymilliliters of Supplement A (bovine serum albumin, 3.2 g/20 mL) and tenmilliliters of Supplement B (ceftazidime pentahydrate, 40 mg/10 mL) weremixed with the coating mixture. The coating mixture with supplements wasknife-coated onto clear polyester film (2.91 mil (0.07 mm) thick) usinga 15 mil (0.38 mm) gap. The coated polyester was dried for about 8minutes in an oven set at 210° C. The final coating weight of the driedcoated mixture was 0.308 g/24 in² (0.308 g/154.8 cm²). Styrofoam spacers(20 mil (0.51 mm) thick), with 2-inch (5.08 cm) diameter openings, wereadhered to the coated, dried film, as described in Example 1 of U.S.Pat. No. 5,601,998; which is incorporated herein by reference in itsentirety. The substrate was cut into approximately 4″ (10.2 cm) by 4″(10.2 cm) pieces, each piece having a spacer with the 5-cm opening thatframed a circular area of the dried, coated broth composition.

TABLE 5 Composition of coating mixture. Component Material Grams  1Proteose peptone No. 3 3.127  2 Casein peptone type III 9.6  3 Casaminoacids 6.0  4 Meat peptone (porcine) 5.0  5 Glucose 2.5  6 Yeast extract7.0  7 Potassium phosphate, dibasic 4.5  8 Lithium chloride 9.0 10 Guargum 12 11 Nalidixic acid, sodium salt 0.01

Preparation of Thin Film Culture Device—Powder-Coated Substrate:

Polyethylene-coated paper (0.13 mm thick) was obtained from SchoellerPaper (Pulaski, N.Y.). 6-Chloro-3-indoxyl-β-D-glucopyranoside (X-gluc,part number B5020) was obtained from Biosynth AG (Staad, Switzerland).The X-gluc (139.1 mg was thoroughly mixed into 200 grams of adhesive (acopolymer of isooctylacrylate and acrylamide in a 96:4 weight ratio).The adhesive mixture was then knife-coated onto the polyethylene-coatedpaper and heated at in an oven at 210° C. to achieve a dry coatingweight of 0.237 g/24 in² (0.237 g/154.8 cm²). The adhesive-coated filmwas coated with a layer of guar gum that was previously disinfectedusing ethylene oxide. The excess guar gum was shaken off and thepowder-coated substrate was cut into approximately 4″ (10.2 cm) by 4″(10.2 cm) pieces.

Assembly of the Thin Film Culture Devices:

A piece of double-stick tape (3M Company, St. Paul, Minn.) was appliedalong one edge of the Styrofoam spacer on each broth-coated substrate. Apowder-coated substrate was aligned (with the powder-coated side facingthe spacer) to superimpose the broth-coated substrate and thepowder-coated side was adhered to the double-stick tape to form theassembled culture devices.

Preparation of Detection Articles:

A coating mixture was prepared by blending into 1000 milliliters ofdeionized water the following components: 13.0 grams of guar gum and asolution of 141.3 milligrams of5-bromo-4-chloro-3-indoxyl-β-myo-inositol-1-phosphate (X-IP, part numberB7404-P00, obtained from Biosynth AG) dissolved in 2.0 mLdimethylsulfoxide (DMSO). The mixture was heated in a covered beaker to80° C. while mixing, mixed at that temperature for an additional 15minutes, and then cooled in a refrigerator. The mixture was warmed toroom temperature before coating. The mixture was knife-coated (25-mil(0.64 mm) gap) onto the clear polyester film described above. The coatedfilm was dried in an oven at 210° C. to achieve a dry coating weight of0.092 g/24 in² (0.092 g/154.8 cm²). The coated substrate was cut intoapproximately 4″ (10.2 cm) by 4″ (10.2 cm) pieces.

Detection Method.

A pure colony of each of the bacteria shown in Table 6 was inoculatedinto separate tubes of tryptic soy broth containing yeast extract(TSBYE). The tubes were incubated at 35° C. for 18-24 hours. Individualculture devices (prepared as described in this Example) were opened andhydrated by pipetting 1.5 mL of Butterfield's buffer into the circulararea of dehydrated broth defined by the Styrofoam spacer. The culturedevices were closed, bringing the powder-coated paper into contact withthe buffer and spreading the buffer over the entire circular growth areadefined by the spacer. The hydrated plates were allowed to sit at roomtemperature for 1 hour.

Ten microliters of each overnight Listeria culture were inoculated bystreak-plate technique into individual hydrated culture devices and theinoculated culture devices were incubated at 35° C. in a zipper-sealplastic bag for 24 hours. After incubation, the colonies were observedand the colony appearance is described in Table 6.

The incubated plates were opened, causing the hydrated gel containingthe bacterial colonies to remain attached to the polypropylene-coatedpaper substrate. A detection article (described in this Example) wasplaced in the culture device such that the coated side of the detectionarticle faced the hydrated gel containing the colonies. The culturedevice was then closed, causing the coated side of the detection articleto contact the hydrated gel containing the bacterial colonies. Theculture devices were then incubated at 35° C. and observed. Theobservations are reported in Table 6. The results indicate that only theListeria monocytogenes microorganisms reacted with (i.e., hydrolyzed)the enzyme substrate indicator(5-bromo-4-chloro-3-indoxyl-β-myo-inositol-1-phosphate) from thedetection article.

TABLE 6 Observations of culture devices containing Listeriamicroorganisms. Colony Appearance Colony Appearance after AddingMicroorganism in Culture Device¹ Detection Article² Listeriamonocytogenes, Reddish-colored Blue-colored ATCC 19111 Listeria grayi,Reddish-colored Reddish-colored ATCC 19120 Listeria ivanovii,Reddish-colored Reddish-colored ATCC 19119 Listeria seeligeri,Reddish-colored Reddish-colored ATCC 35967 Listeria welshimeri,Reddish-colored Reddish-colored ATCC 35897 Listeria innocuaReddish-colored Reddish-colored ¹Colony appearance after 24 hourincubation ²Colony appearance within 15-60 minutes after detectionarticle was contacted with the growth area of the culture device.

Various modifications may be made without departing from the spirit andscope of the invention. These and other embodiments are within the scopeof the following claims.

The invention claimed is:
 1. A method for detecting a Listeriamicroorganism in a sample, the method comprising: providing: a culturedevice including a culture medium comprising ingredients selected tofacilitate growth of a Listeria microorganism; a detection articlecomprising a first indicator system, the first indicator system selectedto detect Listeria monocytogenes; wherein the Listeria microorganism isdetected by its ability to convert the first indicator system from afirst state to a second state; a second indicator system selected todetect the Listeria microorganism; and a sample; inoculating the culturedevice with the sample; placing the second indicator system in fluidcommunication with the culture medium; incubating the inoculated culturedevice for a period of time sufficient to permit growth of the Listeriamicroorganism, if the Listeria microorganism is present in the sample;observing the culture device to detect at least one Listeriamicroorganism; wherein observing the culture device for an indication ofthe presence of the at least one Listeria microorganism comprisesdetecting a conversion of the second indicator system from a first stateto a second state; contacting the culture medium of the incubatedculture device with the first indicator system of the detection articleonly if the at least one Listeria microorganism is detected; andobserving the article-contacted culture device to detect a conversion ofthe first indicator system from a first state to a second state by theat least one Listeria microorganism; wherein the first and secondindicator systems are distinct; wherein conversion of the firstindicator system from the first state to the second state by the atleast one Listeria microorganism indicates the at least oneListeria-microorganism is Listeria monocytogenes.
 2. The method of claim1, wherein providing the culture device further comprises providing aculture device that comprises a hydrogel or a dry, cold-water-solublegelling agent.
 3. The method of claim 1, wherein providing a culturedevice further comprises providing a culture device that includes thesecond indicator system.
 4. The method of claim 1, wherein the firstindicator system comprises a reagent to detect α-mannopyranosidaseand/or phosphatidylinositol-specific phospholipase C enzyme activity. 5.The method of claim 1, wherein observing the culture device or thearticle-contacted culture device comprises observing the culture devicevisually.
 6. The method of claim 1, wherein observing the culture deviceor the article-contacted culture device comprises observing the culturedevice using an imaging device.
 7. The method of claim 1, furthercomprising enumerating Listeria microorganism colony-forming units inthe culture device.
 8. The method of claim 1, further comprisingenumerating Listeria monocytogenes microorganism colony-forming units inthe culture device.
 9. The method of claim 1, wherein contacting theculture medium with the detection article further comprises contactingthe culture medium with the detection article at a predeterminedtemperature.